Combined antitumor effects of anti-EGFR variant III CAR-T cell therapy and PD-1 checkpoint blockade on glioblastoma in mouse model.

Glioblastoma is one of the deadliest cancers. Chimeric antigen receptor (CAR)-T cell therapy against solid tumors has been far from satisfactory largely due to the immunosuppressive tumor microenvironment, such as PD-1 mediated T cell exhaustion. In the present study, we investigated the combined antitumor effects of anti-EGFR variant III CAR-T cell therapy and PD-1 checkpoint blockade on glioblastoma in mouse model. The results demonstrated that CAR-T cells with PD-1 blockade exhibit higher killing efficiency in vitro. Additionally, CAR-T cells with PD-1 blockade showed more effective and persistent therapeutic effects on glioblastoma and led to significantly increased number of tumor infiltrating lymphocytes (TILs) in the mouse model. In conclusion, PD-1 checkpoint blockade significantly enhanced the antitumor activity of anti-human EGFRvIII CAR-T cells by overcoming TILs exhaustion. The outcomes of the present study provide a novel strategy for improving the potency of CAR-T cell therapies in solid tumors.

Molecular vaccine prepared by fusion of XCL1 to the multi-epitope protein of foot-and-mouth disease virus enhances the specific humoural immune response in cattle.

Targeting antigen to dendritic cells (DCs) is a promising way to manipulate the immune response and to design prophylactic molecular vaccines. In this study, the cattle XCL1, ligand of XCR1, was fused to the type O foot-and-mouth disease virus (FMDV) multi-epitope protein (XCL-OB7) to create a molecular vaccine antigen, and an △XCL-OB7 protein with a mutation in XCL1 was used as the control. XCL-OB7 protein specifically bound to the XCR1 receptor, as detected by flow cytometry. Cattle vaccinated with XCL-OB7 showed a significantly higher antibody response than that to the △XCL-OB7 control (P < 0.05). In contrast, when XCL-OB7 was incorporated with poly (I:C) to prepare the vaccine, the antibody response of the immunized cattle was significantly decreased in this group and was lower than that in the △XCL-OB7 plus poly (I:C) group. The FMDV challenge indicated that cattle immunized with the XCL-OB7 alone or the △XCL-OB7 plus poly (I:C) obtained an 80% (4/5) clinical protective rate. However, cattle vaccinated with △XCL-OB7 plus poly (I:C) showed more effective inhibition of virus replication than that in the XCL-OB7 group after viral challenge, according to the presence of antibodies against FMDV non-structural protein 3B. This is the first test of DC-targeted vaccines in veterinary medicine to use XCL1 fused to FMDV antigens. This primary result showed that an XCL1-based molecular vaccine enhanced the antibody response in cattle. This knowledge should be valuable for the development of antibody-dependent vaccines for some infectious diseases in cattle.

The Identification and Distribution of Cattle XCR1 and XCL1 among Peripheral Blood Cells: New Insights into the Design of Dendritic Cells Targeted Veterinary Vaccine.

The chemokine (C motif) receptor 1 (XCR1) and its ligandXCL1 have been intensively studied in the mouse and human immune systems. Here, we determined the molecular characteristics of cattle XCR1 and XCL1 and their distribution among peripheral blood cells. Cattle XCR1 mRNA expression was mainly restricted to CD26+CADM1+CD205+MHCII+CD11b- cells in blood that were otherwise lineage marker negative (lin-); these represented a subset of classic dendritic cells (DCs), not plasmacytoid DCs. Some of these DCs expressed CD11a, CD44, CD80 and CD86, but they did not express CD4, CD8, CD163 or CD172a. Cattle XCL1 was expressed in quiescent NK cells and in activated CD8+ T cells. Cattle XCR1+ DCs migrated chemotactically in response to mouse, but not to human, XCL1. The distribution characters of cattle XCR1 and XCL1 suggested a vital role in regulation of acquired immune responses and indicated a potential for a DC targeted veterinary vaccine in cattle using XCL1 fused antigens.

Elucidating the biosynthetic and regulatory mechanisms of flavonoid-derived bioactive components in Epimedium sagittatum.

Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. In Epimedium, flavonoids have been demonstrated to be the main bioactive components (BCs). However, the molecular biosynthetic and regulatory mechanisms of flavonoid-derived BCs remain obscure. In this study, we isolated 12 structural genes and two putative transcription factors (TFs) in the flavonoid pathway. Phytochemical analysis showed that the total content of four representative BCs (epimedin A, B, C, and icariin) decreased slightly or dramatically in two lines of Epimedium sagittatum during leaf development. Transcriptional analysis revealed that two R2R3-MYB TFs (EsMYBA1 and EsMYBF1), together with a bHLH TF (EsGL3) and WD40 protein (EsTTG1), were supposed to coordinately regulate the anthocyanin and flavonol-derived BCs biosynthesis in leaves. Overexpression of EsFLS (flavonol synthase) in tobacco resulted in increased flavonols content and decreased anthocyanins content in flowers. Moreover, EsMYB12 negatively correlated with the accumulation of the four BCs, and might act as a transcriptional repressor in the flavonoid pathway. Therefore, the anthocyanin pathway may coordinate with the flavonol-derived BCs pathway in Epimedium leaves. A better understanding of the flavonoid biosynthetic and regulatory mechanisms in E. sagittatum will facilitate functional characterization, metabolic engineering, and molecular breeding studies of Epimedium species.

Chemotypic and genetic diversity in Epimedium sagittatum from different geographical regions of China.

Different geographical plant populations within a single species can exhibit variation, in the production of secondary metabolites. Genetic and environmental variations both contribute to differences between populations; however, the relative importance of these factors is unclear. Here, the extent of variation in the production of four flavonoid glycosides (epimedin A, B, C and icariin) were investigated in eleven wild populations of Epimedium sagittatum used in traditional Chinese medicine. Secondary metabolite profiles were classified into five chemotypes. A common garden experiment indicated this chemotype variation has a significant genetic basis. Extensive genetic variation among intraspecific populations was shown using a retrotransposon-based molecular marker system. These results will assist in development of strategies for conservation, utilization and domestication of E. sagittatum.

Variation of medicinal components in a unique geographical accession of horny goat weed Epimedium sagittatum Maxim. (Berberidaceae).

Herbal Epimedium species have been widely in Traditional Chinese Medicine for sexual enhancement, immunity improvement, anticancer and anti-aging treatment, with flavonoids and polysaccharides being the major active components. However, exhaustive depletion of wild sources warrants germplasm evaluation and quality resource exploration. A preliminarily analysis had previously indicated that a specific local geographic accession of Epimedium sagittatum found in Luotian (LT) county of Hubei Province (China) had a much higher content of total flavonoids and polysaccharides. In this study, we further investigated the medicinal component variation in the LT type under different light intensities and in different regions by the common-garden experiment. The results indicated a light intensity range of 40-160 µmol/m²/s was the most suitable for the synthesis and accumulation of total flavonoids, while polysaccharide accumulation was negatively correlated with the light intensity. Icariin was the component displaying the highest content among flavonoids, and the content of major flavonoid bioactive components was relatively stable in the third year after cultivation. There was significant correlation between the major flavonol glycoside constituents and the geographic location, and Central China followed by Northern China were the highly suitable regions for cultivation of LT type E. sagittatum. The results revealed that there was a functional balance between flavonoids and polysaccharides at different developmental stages, and the best harvesting stage should consider the primary contents of interest. This study provides important information on the exploration of quality resources, further breeding approaches and cultivation practices of E. sagittatum, and thus the important insights to enhance our understanding of quality control of traditional medicinal plants.